The long term objectives of this project are to understand the roles of PU.1, a member of the ets family of transcription factors, during B cell differentiation. The studies proposed here are focused on establishing the mechanism of protein-protein interactions of PU.1 in the immunoglobulin kappa 3'-enhancer (kappaE3'-enhancer) and to determine the significance of those interactions on B cell development by generating mutant chimeric animals. We have shown that PU.1 binds to the kappaE3'-enhancer and recruits and binding of a second lymphoid specific transaction factor NF-EM5 (PIP) through protein-protein interaction. This interaction is mediated through the PEST domain of PU.1 and requires phosphorylation at serine 148. Recent studies suggest that both PU.1 and PIP functions synergistically with c-fos plus c-Jun to activate the kappaE3'-enhancer. This synergy can be induced by mitogen activated protein kinase (MAPK) signal pathway. In addition, recently, by complementing PU.1-/-ES cells with various PU.1 deletion proteins, we have identified functional sequences of PU.1 that are important for macrophage development in vitro. The goals of the proposed work are two fold. First, we will determine the mechanism of protein-protein interaction between PU.1 plus PIP and c-FOS plus c-Jun in the kappaE3'-enhancer. We will also study whether the induction of functional synergy between PU.1 plus PIP and c-Fos plus c-Jun by MAPK signal pathway regulates the activity of the kappaE3'- enhancer during B cell differentiation. Second, we will establish ES cell clones in which the endogenous PU.1 gene is replaces with various mutants of PU.1 by homologous recombination. This include the PU.1 mutants that fail to interact with PIP as well as c-Fos plus c-Jun. Additionally, the mutants that have been demonstrated to block macrophage development will also be used as well. Using these knock-in ES cells, we will establish chimeric mice to establish the effect of PU.1 mutations on B lymphocyte development and on the genes (Rag-1, Rag- 2, lambda5, mb-1, B29, IgH, kappa) and regulatory factors (E12/47, Pax- 5, EBF, PIP) that are involved in B cell differentiation. The combined biochemical and genetic experiments proposed here will provide a better understanding of the function of PU.1 which will help in understanding its regulatory roles during hematopoiesis.